The initiating event of many myocardial infarctions (heart attacks) is hemorrhage into an atherosclerotic plaque. Such hemorrhage may result in formation of a thrombus or blood clot in the coronary artery which supplies the infarct zone (i.e., an area of necrosis which results from an obstruction of blood circulation). This thrombus is composed of a combination of fibrin and blood platelets. The formation of a fibrin-platelet clot has sedous clinical ramifications. The degree and duration of the occlusion caused by the fibrin-platelet clot determines the mass of the infarct zone and the extent of damage.
The goal of current treatment for myocardial infarction is the rapid dissolution of the occluding thrombus (thrombolysis) and the restoration of normal blood flow (reperfusion). Successful therapy should include elimination of the fibrin-platelet clot and prevention of its reformation. If the fibrin-platelet clot reforms, then the affected artery may become reoccluded, reversing the benefits of thrombolytic therapy.
The formation of fibrin-platelet clots in other parts of the circulatory system may be partially prevented through the use of anti-coagulants such as heparin. Unfortunately, heparin therapy has not been found to be universally effective in preventing reocclusion in myocardial infarction victims where the degree of blood vessel occlusion (the degree of stenosis) is greater than or equal to about 70%, especially in those patients with severe residual coronary stenosis.
If an individual has formed a fibdn-platelet clot, the clot may be dissolved through the use of a thrombolytic agent. A thrombolytic agent is a medicament capable of lysing the fibrin-platelet thrombus, and thereby permitting blood to again flow through the affected blood vessel. Such agents include; streptokinase, streptokinaseanalogues, prourokinase, urokinase, and tissue-type plasminogen activator (t-PA) (Ganz, W. et al., J. Amer. Coll. Cardiol. 1:1247-1253 [1983]; Rentrop, K. P. et al., Amer. J. Cardiol., 54:29E-31E [1984]; Gold, H. K. et al., Amer. J. Cardiol., 53:22C-125C [1984]).
Treatment with thrombolytic agents can often successfully restore coronary blood flow rapidly enough to interrupt myocardial infarction. Unfortunately, the dissolved fibdn-platelet clot has been found in a number of patients to reform after cessation of such thrombolytic therapy. This reformation may result in the reocclusion of the affected blood vessels, and is, therefore, of substantial concern (Gold, H. K. et al., supra; Gold H. K. et al., Circulation, 68:150-154 [1983]). Thus, although streptokinase treatment has been found to be successful in dissolving fibrin clots, reocclusion of the affected vessels has been found to occur in approximately 25% of the patients examined (Gold, H. K., et al., Circulation 68:150-154 [1983]).
Tissue-type plasminogen activator is believed to be a more desirable thrombolytic agent than either streptokinase or urokinase because it displays greater (though not absolute) specificity for fibrin than either of these latter agents (Verstrate, M., et al., Lancet 1:142 [1985]). Tissue-type plasminogen activator t-PA also displays a rapid clearing rate from plasma. t-PA has been found to be an effective thrombolytic agent in patients with acute myocardial infarction, producing coronary reflow (i.e., decreasing stenosis) in 45-75 minutes in approximately 70% of patients studied (Gold, H. K. et al., Circulation 73:347-352 [1986]).
Typically, tissue-type plasminogen activator is administered as an infusion at a rate of approximately 100 mg/patient. The benefit of employing t-PA is significantly offset by the spontaneous rate of acute reocclusion which follows the cessation of t-PA therapy. It has been observed that cessation of t-PA therapy resulted in reocclusion of affected blood vessels in approximately 45% of patients studied (Circulation 73:347-352 [1986]). Increased t-PA dosages have not been found to decrease the tendency for coronary artery reocclusion. Significantly, the possibility of thrombin clot reformation is closely related to the degree of residual coronary stenosis (i.e., the extent of blood vessel blockage). Thus, reocclusion is more probable in individuals in which high grade stenosis (i.e., greater than 70% quantitative stenosis or greater than 80% non-quantitative stenosis) has occurred. The reocclusion of blood vessels has been found to be inhibited by continued infusion of t-PA (Gold, H. K. et al., Circulation 73:347-352 [1986]).
The general mechanism of blood clot formation is reviewed by Ganong, W. F. (In: Review of Medical Physiology, 13th ed., Lange, Los Altos, Calif., pp 411-414 [1987]). Blood coagulation requires the confluence of two processes; the production of thrombin which induces platelet aggregation and the formation of fibrin which renders the platelet plug stable. A number of discrete proenzymes and procofactors, referred to as coagulation factors, participate in the coagulation process. The process comprises several stages, each requiring separate coagulation factor and ends in fibrin cross-linking and thrombus formation. Briefly, fibrinogen is converted to fibrin by the action of thrombin. Thrombin, in turn, is formed by the proteolytic cleavage of a proenzyme, prothrombin. This proteolysis is effected by activated factor X (factor X.sub.a) which binds to the surface of activated platelets and, in the presence of factor V.sub.a and calcium ion cleaves prothrombin.
Activation of factor X may occur by either of two separate pathways, referred to as the extrinsic and intrinsic pathways. The intrinsic pathway or cascade consists of a series of reactions in which a protein precursor is cleaved to form an active protease. At each stage, the newly formed protease will catalyze the activation of another protease used in a subsequent step of the cascade. Thus the protein product (active protease) of each step acts as a catalyst for the next step. Such a cascade results in a significant amplification of the thrombus forming. A deficiency of any of the proteins in the cascade blocks the activation process at that step, thereby inhibiting or preventing clot formation. Deficiencies of factor VIII or factor IX, for example, are known to cause the bleeding syndromes haemophilia A and B, respectively. In the extrinsic pathway of blood coagulation, tissue factor, also referred to as tissue thromboplastin, is released from damaged cells and facilitates factor X activation in the presence of factor VII and calcium. Although activation of factor X was originally believed to be the only reaction catalyzed by tissue factor and factor VII, it is now known that an amplification loop exists between factor X, factor VII, and factor IX (Osterud, B., and S. I. Rapaport, Proc. Natl. Acad. Sci. USA 74:5260-5264, 1977 and Zur, M. et al., Blood 52:198, [1978]). Each of the sedne proteases in this scheme is capable of converting, by proteolysis, the other two into the activated form, thereby amplifying the signal at this stage in the coagulation process. It is now believed that the extrinsic pathway may in fact be the major physiological pathway of normal blood coagulation (Haemostasis 13:150-155 [1983]). Since tissue factor is not normally found in the blood, the system does not continuously clot; the trigger for coagulation would therefore be the release or exposure of tissue factor from damaged tissue, e.g. atherosclerotic plaque.
Tissue factor is an integral membrane glycoprotein which, as discussed above, can trigger blood coagulation via the extrinsic pathway. (Bach, R. et al., J. Biol Chem. 256(16): 8324-8331 [1981]). Tissue factor consists of a protein component (previously referred to as tissue factor apoprotein-III) and a phospholipid. (Osterud, B. and Rapaport, S. I., PNAS, 74:5260-5264 [1977]). The complex has been found on the membranes of monocytes and other cells of the blood vessel wall. (Osterud, B., Scand. J. Haematol. 32:337-345 [1984]). Recent characterization of tissue factor protein reveals the protein to have a molecular weight of approximately 30,000 daltons. Three asparginine-linked carbohydrate structures increase the apparent molecular weight (SDS-PAGE) to about 45,000 daltons (Bach, R. CRC. Crit. Rev. Biochem., 23:339-368 [1988]). Human tissue thromboplastin has been described as consisting of a tissue factor protein inserted into phospholipid bilayer in an optimal ratio of tissue factor protein: phospholipid of approximately 1:80 (Lyberg, T. and Prydz, H., Nouv. Rev. Fr. Hematol. 25(5):291-293 [1983]). Purification of tissue factor has been reported from various tissues such as human brain (Guha, A. et al. PNAS 83:299-302 [1986] and Broze, G. H. et al., J. Biol. Chem. 260:10917-10920 [1985]), bovine brain (Bach, R. et al., J. Biol. Chem. 256:8324-8331 [1981]), human placenta (Born, V. J. J. et al., i Thrombosis Res. 42:635-643 [1986], and, Andoh, K. et al., Thrombosis Res. 43:275-286 [1986]), ovine brain (Carlsen, E. et al., Thromb. Haemostas. 48(3):315-319 [1982]), and lung (Glas, P. and Astrup, T., Am. J. Physiol., 219:1140-1146 [1970]). It has been shown that bovine and human tissue thromboplastin are identical in size and function. (See for example Broze, G., et al., J. Biol. Chem. 260(20):10917-10920 [1985]). It is widely accepted that while there are differences in structure of tissue factor protein between species there are no functional differences as measured by in vitro coagulation assays. Furthermore, tissue factor isolated from various tissues of an animal, e.g. dog brain, lung, arteries and vein has been shown to be similar in certain respects such as, extinction coefficient, content of nitrogen and phosphorous and optimum phospholipid to lipid ratio but different slightly in molecular size, amino acid content, reactivity with antibody and plasma half life (Gonmori, H. and Takeda, Y., J. Physiol. 229(3):618-626 [1975]). It is also widely accepted that in order to demonstrate biological activity, tissue factor must be associated with phospholipids (Pitlick, F. A. et al., Biochemistry 9:5105-5111 [1970] and Bach, R. et al. supra. at 8324). It has been shown that the removal of the phospholipid component of tissue factor, for example by use of a phospholipase, results in a loss of its biological activity (Nemerson, Y., J. C. I. 47:72-80 [1968]). Relipidation can restore in vitro tissue factor activity. Pitlick, F. A. supra, and Freyssinet, J. M. et al., Thrombosis and Haemostasis 55:112-118 [1986]).
Infusion of tissue factor has long been believed to compromise normal haemostasis. In 1834 the French physiologist de Blainville first established that tissue factor contributed directly to blood coagulation (de Blainville, H. Gazette Medicale Paris, Series 2, 524 [1834]). de Blainville also observed that intravenous infusion of a brain tissue suspension caused immediate death which he observed was correlated with a hypercoagulative state giving rise to extensively disseminated blood clots found on autopsy. It is now well accepted that intravenous infusion of tissue thromboplastin induces intravascular coagulation and may cause death in various animals (dogs: Lewis, J. and Szeto I. F., J. Lab. Clin. Med. 60:261-273 (1962); rabbits: Fedder, G. et al., Thromb. Diath. Haemorrh. 27:365-376 (1972); rats: Giercksky, K. E. et al., Scand. J. Haematol. 17:305-311 (1976); and, sheep: Carlsen, E. et al., Thromb. Haemostas. 48:315-319 [1982]).
In addition to intravascular coagulation or a hypercoagulative state resulting from the exogenous administration of tissue factor, it has been suggested that the intravascular release of tissue thromboplastin may initiate a coagulopathic response such as disseminated intravascular coagulation (DIC) (Prentice, C. R., Clin. Haematol. 14(2):413-442 [1985]). DIC may arise in various conditions such as shock, septicaemia, cardiac arrest, extensive trauma, bites of poisonous snakes, acute liver disease, major surgery, burns, septic abortion, heat stroke, disseminated malignancy, pancreatic and ovarian carcinoma, promyelocytic leukemia, myocardial infarction, neoplasms, systemic lupus erythematosus, renal disease and eclampsia. Thus, the coagulopathic response is known to be associated with vadous disease states including but not limited to septic shock. How the coagulopathic state is induced during septic shock is not known with certainty, but it probably involves alterations in procoagulant, anticoagulant, and/or fibdnolytic properties of endothelial cells as well as other cells within the vasculature (Taylor et al., Circulatory Shock 33:127-134 [1991]). The cause of septic shock can not always be determined, however, it is frequently associated with gram-negative bacterial infection. Gram-negative bacteremia poses a major health problem, causing one-half of cases of lethal septic shock acquired during hospitalization. Bacterial lipopolysaccharide (LPS) and the inflammatory cytokines; tumor necrosis factor (TNF); and interleukin-1 (IL-1), have been shown to be essential mediators of septic shock. Among the effects of these mediators is a coagulopathy that may be triggered by induced expression of tissue factor (TF) on macrophages and endothelial cells. As described above, tissue factor is known to be a potent initiator of the coagulation cascade. Lipopolysaccharide (LPS) from E. coli can both directly and indirectly induce monocytes and macrophages to express TF, as well as to secrete inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor a (TNF.alpha.)(Old, Science, 8:630-632 [1985]). LPS can also induce TF in endothelial cells (Colucci et al., J. Clin. Invest., 71:1893-1896 [1983]). IL-1 and TNF.alpha., which are major mediators of shock in gram-negative sepsis (Tracey et al., Nature (London), 330:662-664 [1984]) in turn can induce expression of TF by endothelial cells cultured in vitro. It is therefore probable that induced expression of TF of monocytes/macrophages and endothelial cells is responsible for triggering the coagulation cascade during septic shock. Present treatment of DIC includes transfusion of blood and fresh frozen plasma; infusion of hepadn; and removal of formed thrombi.
The foregoing clinical syndromes suggest that endogenous release of tissue factor can result in severe clinical complications (see also Andoh, K. et al., Thromb. Res., 43:275-286 [1986]). Efforts have been made to overcome the thrombotic effect of tissue thromboplastin using the enzyme thromboplastinase and a monoclonal antibody specific for tissue factor. Thromboplastinase is a phospholipase and would presumably cleave the phospholipid portion of tissue factor (Gollub, S. et al., Thromb. Diath. Haemorh. 7:470-479 [1962]). Immunoglobulin G (IgG) or Fab fragments of a monocional antibody against tissue factor administered to baboons as a pretreatment has recently been shown to attenuate coagulopathy and protect against an otherwise lethal dose of E coli. (Taylor, supra).
None of the foregoing references suggest a form of tissue factor protein capable of neutralizing the effect of endogenous TF to prevent or inhibit coagulation. A need for such a protein exists since it would provide an important adjunct to thrombolytic therapy as well as neutralize the hypercoagulative effects of induced or endogenously produced TF.
An object of the present invention is to provide an effective therapy for myocardial infarction which limits necrosis by permitting early reperfusion and by preventing reocclusion.
A further object of this invention is to provide a therapeutic composition for treatment of myocardial infarction and prevention of reformation of fibrin-platelet clots, i.e. reocclusion.
Yet another object of this invention is to provide an anticoagulant therapeutic, that is an antagonist to tissue factor protein, to neutralize the thrombotic effects of endogenous release of tissue thromboplastin which may result in a hypercoagulative state. Particularly, such an anticoagulant, that is an antagonist to tissue factor protein, would neutralize the hypercoagulant effects of endogenously released tissue thromboplastin by competing with tissue factor protein binding to factor VII.